Elevated endothelial dysfunction-related biomarker levels indicate the severity and predict sepsis incidence

This study was conducted to investigate the relationship between serum endothelial dysfunction-related biomarker levels and organ dysfunction severity in septic patients and the predictive value of these levels during sepsis. In total, 105 patients admitted to the Department of Critical Care Medicine were enrolled between September 2020 and November 2021. Serum syndecan-1 and soluble thrombomodulin(sTM) levels were measured by enzyme-linked immunosorbent assay, and clinical and laboratory data were recorded. Enroll patients were divided into the infection (n = 28), septic nonshock (n = 31), and septic shock (n = 46) groups . Serum syndecan-1 (102.84 ± 16.53 vs. 55.38 ± 12.34 ng/ml), and sTM(6.60 ± 1.44 ng/ml vs. 5.23 ± 1.23 ng/ml, P < 0.01) levels were increased in the septic group compared with those in the infection group. Serum syndecan-1 levels were closely positively correlated with serum sTM (rs = 0.712, r2 = 0.507, P < 0.001). Additionally, serum syndecan-1(rs = 0.687, r2 = 0.472, P < 0.001) and sTM levels (rs = 0.6, r2 = 0.36, P < 0.01) levels were significantly positively correlated with the sequential organ failure assessment scores respectively. Syndecan-1 (AUC 0.95 ± 0.02, P < 0.0001) was more valuable for prediction sepsis than was sTM (AUC 0.87 ± 0.04, P < 0.0001). Compared with sTM (AUC 0.88 ± 0.03, P < 0.001), syndecan-1 (AUC 0.95 ± 0.02, P < 0.001) and SOFA score (AUC 0.95 ± 0.02, P < 0.001) were better predictors of septic shock. Serum syndecan-1 and sTM levels were associated with organ dysfunction severity in septic patients, and both were good predictors for early identification of sepsis, particularly in patients undergoing septic shock.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection and is associated with significant morbidity and mortality 1 . The pathogenetic mechanism of sepsis is highly complex, and endothelial dysfunction is an important factor leading to organ dysfunction 2 . Endothelial cells shift toward a proinflammatory, proadhesive, and procoagulant phenotype during sepsis. Moreover, the endothelial cells' functional modifications are initially adaptive but ultimately become harmful leading to multiorgan dysfunction during sepsis 3 .
The glycocalyx is the constituent of the endothelial surface layer, which regulates vascular permeability, adhesion of leukocytes and platelets, shear stress, and inflammatory processes 4 .
Syndecan-1 is a member of the polysaccharide syndecan family, which belongs to the family of transmembrane heparan sulfate proteins in the glycocalyx, and circulating syndecan-1 is a marker of endothelial glycocalyx degradation 5 . Previous studies have shown that syndecan-1, is shed and released into the bloodstream during severe infection, reflecting glycocalyx damage and hence a superficial endothelial disruption 6,7 . Studies have demonstrated that syndecan-1 shedding is associated with both sepsis presence and severity 8,9 . Thrombomodulin (TM), an integral endothelial cell membrane protein, is critical in maintaining vascular thrombus resistance 10 . TM has anticoagulant activity as well as anti-inflammatory and cytoprotective effects 11 . Previous studies have shown that TM is enzymatically cleaved from endothelial cells and released into the bloodstream under direct endothelial cell damage, thereby reflecting profound endothelial dysfunction 12 .
As the innermost layer of the arterial wall, the syndecan-1 is the first protein to be affected 13 . However, soluble thrombomodulin (sTM) is derived from direct damage to endothelial cells, not through secretion, which appears later in the circulating. Syndecan-1 and sTM serve as markers of endothelial cell injury in humans but to varying degrees. Furthermore, circulating syndecan 1 and sTM levels have been independently associated with mortality Statistical analysis. Normally distributed data are expressed as the mean and standard deviation and were compared using Student's t-test or one-way analysis of variance. Non-normally distributed data are presented as the median and interquartile intervals and were compared using the Mann-Whitney U test. Categorical variables were compared using the chi-square test and are recorded as proportions. Correlations between normally distributed data were analyzed using the Pearson method. Spearman correlation analysis was performed on nonnormally distributed data. Statistical analysis was performed using SPSS 23 (SPSS, IBM, Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). Two-tailed P values ≤ 0.05 were considered statistically significant. Sample size was estimated based on a priori power calculation indicating an 80% power to detect differences in syndecan-1 and soluble thrombomodulin(sTM) (effect size 0.7) among groups at a 0.05 significance level using a power and sample size website.
Ethics approval and consent to participate. The study was approved by the PUMCH institutional review board (Ethics Approval No.JS-3283). All subjects provided written informed consent.

Results
Patients' general and clinical characteristics. We screened 131 patients for enrolment, and 105 were included in this study (Fig. 1). The patients were divided into the infection (n = 28), septic nonshock (n = 31), and septic shock (n = 46) groups. Age, sex, venous-to-arterial carbon dioxide difference (Pv-aCO2), central venous blood oxygen saturation (ScvO 2 ), mean arterial blood pressure (MAP), and central venous pressure (CVP) did not significantly differ among the groups. Patients with septic shock had significantly higher SOFA scores and lactate levels than did the septic nonshock group. Table 1 shows the basic characteristics of all enrolled patients.
Serum levels of endothelial damage-related biomarkers in a derivation cohort. Figure 2 and Table 2 show the changes in endothelial damage-related biomarkers in the progression from infection to septic shock. Serum syndecan-1 and sTM levels increased progressively from infected patients to septic nonstock patients to septic shock patients. The syndecan-1 and sTM level variation tendencies were similar among the groups. Serum syndecan-1 concentrations on ICU admission were significantly increased in the septic shock group and septic nonshock groups compared with those of the infection group.
Syndecan-1 levels were significantly increased in the septic shock group (102.84 ± 16.53 ng/ml) compared with those of the septic nonshock (76.06 ± 10.51 ng/ml) and infection (55.38 ± 12.34 ng/ml) groups. Serum sTM concentrations were significantly increased in the septic shock and septic nonshock groups compared with those of the infection group. www.nature.com/scientificreports/ The septic shock group had higher sTM levels than did the septic nonshock group (9.67 ± 3.38 ng/ml vs. 6.60 ± 1.44 ng/ml, P < 0.01), and patients with septic nonshock had higher sTM levels than did patients with infection (6.60 ± 1.44 ng/ml vs. 5.23 ± 1.23 ng/ml, P < 0.01).
However, as result shown, no statistical difference in the serum syndecan-1 and sTM was seen when all patients (including infection ,sepsis non-shock and septic shock patients) were stratified by bacteremia and non-bacteremia (syndecan-1 82.47(61. 14 Serum syndecan-1 and sTM levels were associated with organ function and disease severity. To establish whether serum syndecan-1 and sTM levels could determine organ function and disease severity, we assessed the correlations between these levels and the SOFA and APACHE II scores on ICU admission. Serum syndecan-1 levels were positively correlated with serum sTM (r s = 0.712, r 2 = 0.507, P < 0.001, Fig. 3a), the both serum syndecan-1 (r s = 0.687, r 2 = 0.472, P < 0.001, Fig. 3b) and sTM (r s = 0.6, r 2 = 0.36, P < 0.01, Fig. 3c) levels were positively correlated with SOFA scores respectively. Additionally, serum syndecan-1 levels were weakly associated with APACHE II score (r s = 0.286, r 2 = 0.082, P = 0.0049, Fig. 3d); however, we found no correlation between serum sTM levels and APACHE II score (r = 0.184, r 2 = 0.034, P = 0.073).  Table 1. Patient characteristics at ICU admission. Data was presented by mean standard deviation, n (%), or median (interquartile range). For normally distributed data, comparisons among multiple groups were conducted by one-way analysis of variance, and those between two groups were carried out with the Least Significant Difference (LSD) post hoc test. For data without normal distribution, Post hoc tests and betweengroup analyses were performed by Mann-Whitney nonparametric tests and Bonferroni corrected. APACHE Acute Physiology and Chronic Health Evaluation, SOFA sequential organ failure assessment score, HR heart rate, MAP mean arterial blood pressure, CVP central venous pressure, P v-a CO 2 venous-to-arterial carbon dioxide difference (Pv-aCO2), ScvO 2 central venous blood oxygen saturation. *P < 0.05 for the comparison between infection and septic non-shock, between infection and septic shock. # P < 0.05 for the comparison between septic non-shock and septic shock. www.nature.com/scientificreports/ We further assessed whether serum syndecan-1 and sTM levels were correlated with organ dysfunction at study enrollment and found that neither serum syndecan1 levels nor sTM levels were significantly correlated with oxygen index, total bilirubin, or creatinine (data not shown).
Serum syndecan-1 and sTM levels were associated with tissue perfusion. To assess the relationship between endothelial damage and tissue perfusion, we assessed the association between serum syndecan-1 and sTM levels and lactate (lac). Serum syndecan-1 (r s = 0.574, r 2 = 0.329, P < 0.001, Fig. 4a) and sTM (r s = 0.458, r 2 = 0.210, P < 0.001, Fig. 4b) levels in all patients admitted to the ICU were significantly positively correlated with lactate, demonstrating that endothelial damage was closely related to tissue perfusion (Fig. 4).
Serum syndecan-1 was associated with coagulation function. Endothelial injury induces coagulation abnormalities during sepsis; therefore, we assessed the association between serum syndecan-1 and sTM levels and coagulation function parameters.

Figure 2.
Comparison of serum syndecan-1 levels (a) and sTM (b) levels among groups. Comparisons among multiple groups were conducted by one-way analysis of variance, and those between two groups were carried out with the Least Significant Difference (LSD) post hoc test. P < 0.05 were considered statistically significant. *Denotes P < 0.05, ***Denotes P < 0.001. sTM serum thrombomodulin. Table 2. Clinical characteristics. Data was presented by mean standard deviation. For normally distributed data, comparisons among multiple groups were conducted by one-way analysis of variance, and those between two groups were carried out with the Least Significant Difference (LSD) post hoc test. For data without normal distribution, Post hoc tests and between-group analyses were performed by Mann-Whitney nonparametric tests and Bonferroni corrected. sTM serum thrombomodulin, OI oxygen index, TB total bilirubin, Cr creatinine, PT prothrombin time, APTT activated partial thromboplastin time, INR International Normalized Ratio, PLT platelet. *P < 0.05 for the comparison between infection and septic non-shock, between infection and septic shock. # P < 0.05 for the comparison between septic non-shock and septic shock.

Discussion
Our results indicated the following. First, both syndecan-1 and sTM levels were significantly increased in the septic shock group compared with those of the septic nonshock and the infection groups. The syndecan-1 and sTM levels gradually increased as the disease progressed, suggesting that endothelial injury was gradually aggravated. Syndecan-1 and sTM levels were significantly higher in patients with septic shock than in patients with infection and septic nonshock.
Moreover, serum syndecan-1 and sTM levels were elevated as the disease severity increased. Notably, serum syndecan-1 and sTM levels were positively correlated with both SOFA score and lactate. These results demonstrated the value of syndecan-1 and sTM levels as predictors of sepsis or septic shock incidence on the day of ICU admission.
Endothelial dysfunction is common in adult ICU patients, especially in critical patients with infection. Endothelial injury is a well-known hallmark of sepsis, but new data continue to emerge that further reveal the  www.nature.com/scientificreports/ pathophysiology of endothelial damage in sepsis and its association with disease severity, including the applicability of biomarkers for outcomes 16,17 . However, different studies have yielded varying conclusions. Anand et al. reported significant correlations between syndecan-1 levels and disease severity 18 . Deng et al. found that patients with sepsis had significantly higher serum sTM levels that were positively correlated with the disease severity 19 . Here, we revealed that the serum syndecan-1 and sTM levels were elevated with the disease severity, which is consistent with previous findings, but the molecular mechanism of this elevation remains undefined.
Syndecan-1 is a proteoglycan in the endothelial glycocalyx. Circulating syndecan-1 is a marker of endothelial glycocalyx degradation, which reflects superficial endothelial damage 20 . However, the mechanism underlying the endothelial glycocalyx during sepsis remains unclear. Metalloproteinases are activated in inflammatory states by reactive oxygen species and proinflammatory cytokines, which cleave proteoglycans directly from the endothelial cell membrane during sepsis 4 . The degraded glycocalyx layer leads to an increased permeability to plasma proteins and fluids, causing interstitial leakage. Several studies have demonstrated elevated syndecan-1 levels as a marker of glycocalyx degradation in patients with sepsis 21,22 .
Furthermore, endothelial dysfunction ultimately contributes to ending multiorgan damage during sepsis or septic shock. Considering the close relationship between endothelial function and organ function during sepsis 1 , we also explored the correlation between serum endothelial dysfunction biomarker levels and the SOFA scores and found that serum syndecan-1 and sTM levels were closely related to SOFA scores. Previous studies have shown that plasma syndecan-1 and sTM increased progressively and significantly across groups with increasing infectious severity and correlated significantly with organ failure as measured by SOFA scores in patients with varying degrees of infectious disease 23 .
Similarly, a large multicenter study of 1103 critically ill patients predominantly suffering from sepsis showed a strong association between epitheliopathy and organ failure 17 , demonstrating that patients with sepsis had higher plasma syndecan-1 and sTM levels than did non-infected patients. These results are consistent with the above idea and support our conclusion. Although the correlation was low in previous studies, our results showed a stronger correlation, which may be related to patient and sample heterogeneity and requires further confirmation.
Previous studies indicated that high syndecan-1 and sTM levels independently predicted liver and renal failure, respectively, and high sTM was further associated with an increased risk of developing multiple organ failure 24 . However, further analysis showed no significant correlations between syndecan-1, sTM, and organ function-related biomarker levels in septic patients. Moreover, endothelial dysfunction shifts toward a proapoptotic, proinflammatory, proadhesive, and procoagulant phenotype during sepsis, thus indicating the central role of endothelial dysfunction in the cross-talk during inflammation coagulation 25,26 . A previous study found an independent association between high circulating syndecan-1 levels and coagulopathy in a smaller cohort of 184 patients with severe sepsis or septic shock 23 .
Nevertheless, the correlations between endothelial dysfunction and coagulation found in the present study were relatively weak; thus, this correlation remains uncertain.
Sepsis-induced organ dysfunction is associated with inflammatory and coagulation responses in the endothelium as described above. Previous studies showed that endothelial dysfunction may result in decreased blood perfusion and therefore may aggravate lactic acidemia 27 . Microvascular obstruction and systemic endothelial dysfunction have been independently associated with plasma lactate in patients with falciparum malaria 28 . Here, we revealed that serum syndecan-1 and sTM levels were significantly correlated with lactate, which was consistent with previous findings, indicating that endothelial damage was closed correlated with tissue perfusion. Perfusion abnormalities caused by endothelial dysfunction may be another important cause of sepsis-related organ failure. Our previous study proposed mitochondrial dysfunction as an important cause of sepsis-related organ failure 29 . Thus, endothelial function induced by mitochondrial dysfunction might be an additional mechanism during sepsis.
Although clinicians know that endothelial activation and dysfunction play critical roles in the pathophysiology of sepsis and represent important therapeutic targets for reducing sepsis mortality, a large gap remains between basic research and clinical applications of endothelial activation and dysfunction. Thus, endothelial damage must be identified early in sepsis or septic shock. Elevated serum syndecan-1 and sTM levels could be used to alert clinicians of endothelial injury, and in clinical treatment, clinicians should put more emphasis on endothelial function and protecting endothelial cells from injury during sepsis.
Limitations. This study has several limitations. First, our sample size was limited, and replication studies with a larger sample size are needed. Second, our findings were observational, hence, no causality can be inferred. Third, the results should be interpreted with caution owing to the heterogeneity of enrolled patients. Fourth, we analyzed only blood taken from the initial draw and did not follow the biomarker dynamics over time. Fifth, we included no healthy control group, and the serum syndecan-1 and sTM levels at baseline need to be measured.

Conclusion
Syndecan-1 and sTM levels were positively associated with organ damage in patients with sepsis and septic shock. Serum syndecan-1 and sTM may be promising biomarkers for early diagnosis of sepsis, particularly for patients with septic shock.

Data availability
Data are available on request from the corresponding author.